桑叶中1-脱氧野尻霉素的提取和对高糖诱导的肾小球系膜细胞凋亡和炎性因子的影响

Extraction of 1-Deoxynojirimycin from Mulberry Leaves and Its Effect on High Glucose-Induced Apoptosis and Inflammatory Factors of Glomerular Mesangial Cells

目的:对桑叶中1-脱氧野尻霉素的提取条件进行优化后研究其对高糖诱导的肾小球系膜细胞凋亡和炎性因子的影响。方法:建立1-脱氧野尻霉素的定量检测方法,探讨桑叶中1-脱氧野尻霉素的最佳提取条件。培养小鼠肾小球系膜(SV40 MES 13)细胞,并分为四组:正常对照组(PBS)、高糖组(30 mmoL/L葡萄糖)和1-脱氧野尻霉素组(2、50μM),给药结束后采用CCK-8法测定SV40 MES 13细胞的存活率,ELISA测定炎性因子单核细胞趋化蛋白1(Monocyte chemoattractant protein-1,MCP-1)、肿瘤坏死因子α(Tumor necrosis factor-alpha,TNF-α)和白细胞介素6(Interleukin-6,IL-6)的水平。最后用Western blotting法检测细胞增殖蛋白Cyclin D1和PCNA的表达水平。结果:桑叶中1-脱氧野尻霉素的提取条件最终优化为料液比1∶15 g/mL、pH值3.0和提取时间30 min。1-脱氧野尻霉素明显提高SV40 MES 13细胞的存活率,并下调炎性因子IL-6的表达水平,且高剂量的1-脱氧野尻霉素显著降低Cyclin D1和PCNA的表达。结论:选择合适的料液比、pH值和提取时间可优化桑叶中1-脱氧野尻霉素的提取工艺,此外,1-脱氧野尻霉素具有改善高糖诱导的肾小球系膜细胞凋亡的作用,其作用机制与调控炎性因子及相关增值蛋白的表达有关。

Objective This project is mainly to optimize the extraction conditions of 1-deoxynojirimycin from mulberry leaves and study its effects on high glucose-induced apoptosis and inflammatory factors of glomerular mesangial cells.Method After establishing the quantitative detection method of 1-deoxynojirimycin,the effects of different solid-liquid ratio,pH value and extraction time on the extraction yield of 1-deoxynojirimycin in mulberry leaves were investigated,and the optimal extraction condition was determined.Mouse mesangial cells(SV40 MES 13)were cultured and divided into four groups:normal control group(PBS),high glucose group(30 mmoL/L glucose)and 1-deoxynojirimycin group(2,50μM).After administration,the survival rate of SV40 MES 13 cells was measured by CCK-8 method.ELISA kits were used to measure the inflammatory cytokines MCP-1,TNF-αand IL-6 levels.The expression levels of Cyclin D1 and PCNA were detected by Western blotting.Results The extraction conditions of 1-deoxynojirimycin from mulberry leaves were finally optimized as solid-liquid ratio of 1:15 g/mL,pH 3.0 and extraction time of 30min.Both low and high dose of 1-deoxynojirimycin significantly increased the survival rate of SV40 MES 13 cells and down-regulated the expression of inflammatory factor IL-6.In addition,high dose of 1-deoxynojirimycin significantly decreased the expression of proliferation-related proteins Cyclin D1 and PCNA.Conclusions The optimal extraction process of 1-deoxynojirimycin from mulberry leaves can be optimized by selecting the appropriate ratio of material to liquid,pH value and extraction time.In addition,1-deoxynojirimycin can significantly improve the apoptosis of glomerular mesangial cells induced by high glucose,and its mechanism is related to the regulation of inflammatory factors and the expression levels of proliferating proteins Cyclin D1 and PCNA.